Structural basis for impaired 5′ processing of a mutant tRNA associated with defects in neuronal homeostasis

Significance Understanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNAArgUCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.


Preparation of n-Tr20 variants and n-Tr21
Sequences of pre-n-Tr20+3' and -n-Tr21 were amplified from C57BL/6NJ genomic DNA using PCR. In each case, primers were designed to include (i) 30-bp upstream flanking sequence for a 5' leader, and (ii) the entire 3' trailer sequence up to the polyT stretch that signals RNA polymerase III transcriptional termination. PCR amplicons were then inserted into the StuI site just downstream of the T7 RNA polymerase promoter in pBT7 (1); note that in vitro transcription (IVT) from this promoter results in two additional Gs at the 5' end of the transcript. The pre-n-Tr20+3' clone was used as the template for PCR-based site-directed mutagenesis to create pre-n-Tr20 derivatives with the C50U, G64A, C50U/G64A, and C56A mutations. Mature n-Tr20 variants were then generated by PCR-based deletion of the 5' leader. All clones were confirmed by automated sequencing at the Genome Resources Facility (OSU Comprehensive Cancer Center). PCR was also used to generate templates from the above clones for run-off IVT with T7 RNA polymerase; the forward primer F-ext anneals upstream of the T7 RNA polymerase promoter while specific reverse primers were used to either include or exclude the 3' trailer.
For multiple-turnover RNase P assays, IVT-generated unlabeled pre-tRNA substrate was spiked with a trace amount (1000 dpm/reaction) of substrate that had been uniformly labeled by IVT with [a-32 P]-GTP and P266L T7 RNA polymerase (2), which yielded a higher labeling efficiency than the wild type. For mapping the RNase P cleavage site and for single-turnover assays, pre-tRNA transcripts were 5'-labeled with [g-32 P]-ATP and T4 polynucleotide kinase (New England Biolabs). In all instances, labeled RNAs were purified by denaturing polyacrylamide gel electrophoresis (PAGE) before use.

Partial purification of RNase P from mouse cortices/livers
Cortices from 4-week-old C57BL/6J (B6J) mice were harvested, flash frozen in liquid nitrogen, and stored at -80°C until use. After multiple pilot trials to determine the best approach for isolating native RNase P from these cortices, the following purification scheme was chosen. A motorized Dounce homogenizer was used to lyse approximately 1.8 g of tissue in 20 mL ice-cold extraction buffer (EB: 20 mM Tris-HCl, pH 8 at 23°C; 5 mM MgCl2; 14 mM b-mercaptoethanol; 0.2 mM PMSF) supplemented with 100 mM NaCl (EB100). Cellular debris was removed by centrifugation at 21,000 x g for 30 min at 4°C. After passing through a 0.45-µm syringe filter, the lysate was loaded onto a 1-mL HiTrap DEAE-FF column (Cytiva) pre-equilibrated with EB100. Using an ÄKTA FPLC (Cytiva), the column was washed with 8 mL EB100 and bound species subsequently eluted using a linear 100-700 mM NaCl (in EB) gradient in 20 x 0.5-mL fractions. Each fraction was then assayed for RNase P activity with pre-n-Tr20 and pre-n-Tr21 ( Fig. 1 A and B) to determine the peaks of activity ( Fig. 1 C and D). Fractions #7-12 were pooled corresponding to peak 1 (P1) and #13-20 to P2 (Fig. 1 C and D). P1 was further purified using a 10-35% (v/v) glycerol gradient (in EB): 1.25 mL P1 was loaded on each of two 10-mL gradients and spun at 200,000 x g for 18 h at 4°C using a Beckman ultracentrifuge. Each gradient was aliquoted from top to bottom into 22 x 0.5-mL fractions. After assaying for RNase P activity (Fig. 1 E and F) with pre-n-Tr20 and pre-n-Tr21, peak fractions #14-16 were pooled and concentrated ~7-fold using Ultra-4 centrifugal filters (3,000 NMWL, Amicon) to yield P1F (final P1). Because further purification (including a glycerol gradient) led to loss of activity, P2 was concentrated ~10-fold to yield P2F.
For purification of RNase P from mouse livers (SI Appendix, Fig. S3), we used the same scheme as that used for the mouse brain except that we only followed the steps until the DEAE-FF elution.

Mouse RNase P assays
DEAE-purified fractions were assayed in EB, 50 nM pre-tRNA, and a trace amount (~1 nM) of uniformly radiolabeled pre-tRNA; the NaCl concentration in each reaction varied (30-210 mM) because each fraction contained a different amount of NaCl due to the gradient employed for elution. For all other multiple-turnover assays, the above reactions were supplemented with 140 mM KCl. All reactions were incubated in either a 37°C water bath or a thermocycler for the indicated period. Time-course reactions were set up in a large volume and aliquots withdrawn at specific time intervals (typically every 5 min). We ensured that total cleavage did not exceed 25% of the substrate. The reactions/aliquots were immediately terminated by adding 1-3 volumes of . Samples were then electrophoresed on 8% (w/v) acrylamide/7 M urea gels, which were subsequently scanned using a Typhoon phosphorimager (GE Healthcare). ImageQuant (GE Healthcare) was used to determine band intensities. The amount of product generated as a function of time was then plotted and the data fit to a straight line in Microsoft Excel to obtain the initial velocity. All linear curve fits had correlation coefficients (R 2 ) ≥ 0.96. For rate determination, all initial velocity measurements were performed when P1F and P2F were less than a week old to ensure that they were highly active. Although all three assay replicates were also completed on the same day to minimize variability ( Fig. 2), we verified that trends were reproducible across independent preparations of mouse brain RNase P.

Thermal denaturation of mature n-Tr20 and its mutant derivatives
One-mL solutions containing 500 nM tRNA in 20 mM sodium cacodylate, pH 7; 150 mM NaCl; and either 0, 1, or 5 mM MgCl2 were prepared and transferred to rectangular semi-micro quartz spectrophotometer cuvettes (FireflySci, Staten Island, NY) with 1-cm path lengths and polytetrafluoroethylene stoppers, which minimized sample loss by evaporation. Because the pKa of cacodylate (~6.3) is independent of temperature, all thermal denaturation profiles were obtained in sodium cacodylate instead of Tris, the buffering agent used during enzyme purification and characterization. Multiple samples were analyzed in parallel using a Cary 3500 ultraviolet (UV)visible double beam, multi-cell Peltier spectrophotometer (Agilent) equipped with eight cuvette positions, which increased experiment throughput and permitted the comparison of samples under identical conditions. Absorbance at 260 nm was monitored as the temperature was increased from 20°C to 95°C at a rate of 0.5°C/min. Tm values were calculated from first-derivative plots (dA/dT vs. T, where A is absorbance and T is temperature) generated using the Analysis function of the Cary UV Workstation Thermal program (Agilent) and a low/high calculation limit of 55-80°C. Mean and standard deviation values were calculated from three independent replicates of each tRNA in each MgCl2 concentration.
Microsoft Excel was used to normalize thermal denaturation curves and to smooth the first derivative (using the moving averages function and an interval of 5), while Kaleidagraph (Synergy Software) was used to plot and overlay the resultant data (Fig. S4).
Although first-derivative analyses of thermal denaturation curves offer an expedient approach to determining Tm values, fitting the data to the van 't Hoff equation allows for independent crossvalidation of these Tm values. Tm values calculated using the first-derivative approach were in good agreement with those obtained using the van 't Hoff equation (3). Note that the tRNAs used in ref. 3 contained a 1-nt leader that was absent in all n-Tr20 variants used here.

Native polyacrylamide gel electrophoresis (PAGE)
Five pmol of mature tRNA was incubated in water at 95°C for 3 min and at 37°C for 10 min before 2 µL 5X buffer [100 mM HEPES, pH 7.5 at 22°C; 100 mM NaCl] and either water or MgCl2 were added to a final volume of 10 µL with to achieve the desired MgCl2 concentration. The sample was then incubated at 37°C for an additional 30 min. Subsequently, 3 µL loading buffer [50% (v/v) glycerol, 0.05% (w/v) bromophenol blue, 0.05% (w/v) xylene cyanol] was added and the samples were kept at 37°C until loading. The same buffer (89 mM Tris, 89 mM boric acid, 2 mM MgCl2) was used to prepare 5% (w/v) polyacrylamide (19:1 acrylamide:bis-acrylamide) gels and used as the running buffer for electrophoresis. Gels (17 cm width x 15 cm height) were electrophoresed at 180 V and 4°C until the bromophenol blue dye reached the bottom. Gels were stained with SYBR Gold (Invitrogen) in TBE buffer for 5 min and then scanned using a Typhoon phosphorimager (GE Healthcare) and the Cy2 imaging mode.

Single-turnover experiments with E. coli RNase P
Since we expected in vitro reconstituted E. coli RNase P to cleave pre-n-Tr20 too rapidly at nearneutral pH, we resorted to an assay pH of 5.5 where the rate is lower (5, 6) and assays can be performed manually. For these single-turnover studies, the in vitro transcribed E. coli RNase P RNA was first refolded. The RNA was incubated in water at 50°C for 30 min and at 37°C for 10 min, followed by addition of an equal volume of 2X binding buffer [2X BB: 40 mM cacodylic acid, 800 mM ammonium acetate, 10 mM magnesium acetate, 10% (v/v) glycerol, 0.02% (v/v) IGEPAL; the solution was pH-adjusted to 5.5 with HCl] and an incubation at 37°C for 30 min. The refolded RNA was then mixed with E. coli RNase P protein (final RNA:protein ratio of 1:2.5) and the mixture 7 was incubated at 37°C for 5 min to promote assembly. To determine the kobs at 37°C, 2000 dpm of 5'-radiolabeled pre-n-Tr20 or pre-n-Tr20 C50U (∼1 fM) was added to the reaction (without   S1. Mouse brain RNase P cleaves both pre-n-Tr20 and pre-n-Tr20 C50U at the correct site.

RNase P
P1F and P2F, the two peaks of mouse brain RNase P activity; T1, a ladder generated by partial digestion of denatured pre-n-Tr20 with RNase T1, a nuclease that cleaves 3' to G residues.
Following RNase T1 digestion, cleavage products were treated with T4 polynucleotide kinase to remove their 3'-phosphate and normalize their migration with the RNase P-cleaved 5' leader that contains a 3'-OH. The 5' leader sequence and the RNase P cleavage site (arrow) are provided to the left of the gel image, with G nucleotides matched to those observed in the T1 ladder. Note that while the 5' leaders of both pre-n-Tr20 and pre-n-Tr20 C50U cleaved by P1F and P2F match the correct product (i.e., cleavage between G-1 and G+1), the leader generated by E. coli RNase P corresponds to mis-cleavage between G-1 and A-2, likely because pre-n-Tr20 and pre-n-Tr20 C50U have a G-1 (a negative determinant) and lack a 3'-CCA (a positive determinant for bacterial RNase P) (9). This mis-cleavage (i.e., single-nt shift) by E. coli RNase P is evident only when employing long gels such as the one shown here and not in any of the assay gels used in this study.   S3. Pre-n-Tr20 C50U is also poorly processed by mouse liver RNase P in 1 or 5 mM MgCl2.
The enzyme was purified using DEAE (anion-exchange) chromatography and one fraction within peak 1 (P1) was assayed. Lanes: -, negative control containing substrate but no enzyme; +, positive control with substrate and in vitro reconstituted E. coli RNase P (8); ML, mouse liver RNase P.     (middle), and n-Tr20 C56A (bottom) are superimposed on n-Tr20 (left) and n-Tr20 C50U (right). Note the similarity between n-Tr20 and n-Tr20 C56A and between n-Tr20 C50U and n-Tr20 C50U/G64A , as well as the distinctive features of n-Tr20 G64A . Black arrows indicate the two signature upfield peaks that are likely associated with tertiary interactions.